Anthranilate synthase (AS) catalyzes the first reaction branching from the aromatic amino acid pathway to the biosynthesis of tryptophan in plants, fungi, and bacteria. The reaction catalyzed by anthranilate synthase is the conversion of chorismate to anthranilate in a glutamine-dependent reaction. In microorganisms, anthranilate synthase is composed of two non-identical subunits: the alpha subunit binds chorismate and eliminates the enolpyruvate side chain, and the beta subunit transfers an amino group from glutamine to the position vacated by the enolpyruvate moiety.
The next reaction in the synthesis of tryptophan is the transfer of the phosphoribosyl moiety of phosphoribosyl pyrophosphate to anthranilate. The indole ring is formed in two steps involving an isomerization converting the ribose group to a ribulose followed by a cyclization reaction to yield indole glycerol phosphate. The final reaction in the pathway is catalyzed by a single enzyme that may contain either one or two subunits. The reaction accomplishes the cleavage of indole glyceraldehyde-3-phosphate and condensation of the indole group with serine (Umbarger, Ann. Rev. Biochem, 47, 555 (1978)).
Metabolite flow in the tryptophan pathway in higher plants and microorganisms is apparently regulated through feedback inhibition of anthranilate synthase by tryptophan. Thus, because anthranilate synthase is feedback inhibited by tryptophan, the overproduction of wild-type anthranilate synthase cannot result in tryptophan overproduction.
While anthranilate synthase has been partially purified from crude extracts of cell cultures of higher plants (Hankins et al., Plant Physiol., 57, 101 (1976); Widholm, Biochim. Biophys. Acta, 320, 217 (1973)), it was found to be very unstable. In order to fUrther characterize the anthranilate synthase of plants, Niyogi and Fink (Plant Cell, 4, 721 (1992)) and Niyogi et al. (Plant Cell, 5, 1011 (1993)) employed a molecular approach. They found that Arabidopsis anthranilate synthase alpha subunits are encoded by two closely related, nonallelic genes which are differentially regulated. One of these alpha subunit genes, ASA1, is induced by wounding and bacterial pathogen infiltration, implicating its involvement in a defense response, whereas the other alpha subunit gene, ASA2, is expressed at constitutive basal levels. Both predicted proteins share regions of homology with bacterial and fungal anthranilate synthase proteins, and contain conserved amino acid residues at positions that have been shown to be involved in tryptophan feedback inhibition in bacteria (Caligiuri et al., J. Biol. Chem., 266, 8328 (1991)).
Amino acid analogs of tryptophan or of intermediates in the tryptophan biosynthetic pathway (e.g., 5-methyltryptophan, 4-methyltryptophan, 5-fluorotryptophan, 5-hydroxytryptophan, 7-azatryptophan, 3xcex2-indoleacrylic acid, 3-methylanthranilic acid) have been shown to inhibit the growth of both prokaryotic and eukaryotic cultures. Plant cell cultures can be selected for resistance to these amino acid analogs. For example, cultured tobacco, carrot, potato, corn and Datura innoxia cell lines have been selected which are resistant to growth inhibition by 5-methyltryptophan (5-MT), an amino acid analog of tryptophan, due to expression of an altered anthranilate synthase as described below.
Widhohn (Biochem. Biophys. Acta, 261, 44 (1972)) demonstrated that the tryptophan analogs 5-MT, 4-methyltryptophan, 5-fluorotryptophan and 6-fluorotryptophan cause growth inhibition of tobacco (Nicotiana tabacum) and carrot (Daucus carota) cell cultures. This inhibition of growth could be reversed by the addition of anthranilic acid, indole, or L-tryptophan. Anthranilate synthase was determined to be very sensitive to these analogs. The tryptophan analogs inhibited cell growth by limiting tryptophan synthesis through the inhibition of anthranilate synthase.
While growth of many cultured tobacco cell lines was inhibited by 5-MT, some tobacco cell lines were resistant to growth inhibitory concentrations of 5-MT (Widholm, Biochim. Biophys. Acta, 261, 52 (1972)). The resistant phenotype was stable for at least 60 cell mass doublings even without selection pressure (i.e., without 5-MT). In addition, 5-MT resistant cells were resistant to growth inhibition by other tryptophan analogs. Free tryptophan levels were increased in 5-MT resistant cells about 10-fold over control tissue. Anthranilate synthase in these 5-MT resistant cells was found to be less sensitive to inhibition by tryptophan or 5-MT.
Carrot cell lines that were resistant to growth inhibition by 5-MT were also selected by Widholm (Biochim. Biophys. Acta, 279, 48 (1972)). This phenotype was generally stable in the absence of the tryptophan analog for at least 100 cell doublings. 5-MT resistant cells were also resistant to other tryptophan analogs. Free tryptophan concentrations in 5-MT resistant cells were substantially increased to 2170 xcexcM as compared to 81 xcexcM (27-fold) for control tissue. Anthranilate synthase was shown to be altered in the 5-MT resistant cells. The enzyme was about 5-fold less sensitive to inhibition by tryptophan or 5-MT than an unaltered anthranilate synthase.
Singh et al. (Biochem. Genet., 13, 357 (1975)) described a mutant in corn, Zea mays L., blue fluorescent-1, that possessed increased anthranilate synthase activity which was less sensitive to feedback inhibition. The mutant also accumulated anthranilic acid. In contrast to previous work in tobacco and carrot, however, the altered anthranilate synthase activity did not lead to significant overproduction of tryptophan in mature corn plants or seed.
Hibberd et al. (U.S. Pat. No. 4,581,847, issued Apr. 15, 1986) described 5-MT resistant maize cell lines that contained an anthranilate synthase that was less sensitive to feedback inhibition than wild-type anthranilate synthase. One 5-MT resistant cell line accumulated free tryptophan at levels almost twenty-fold greater than that of non-transformed cell lines.
Carlson et al. (Physiol. Plant, 44, 251 (1978)) obtained potato cell (Solanum tuberosum) cultures resistant to 5-MT. Anthranilate synthase in these cultures was shown to be less sensitive to inhibition by tryptophan or by 5-MT, although both 5-MT resistant and sensitive forms of the enzyme were present in the cells of the culture. In the selected cell lines, the level of resistant anthranilate synthase was greatly increased relative to the level of the sensitive form. The range of free tryptophan concentrations in selected cultures was from 970 to 1400 xcexcM compared to control cultures in which the tryptophan concentrations were about 29 xcexcM.
Widholm (Plant Cell Cultures: Results and Perspectives, F. Sala, B. Parisi, R. Cella, O. Ciferri (eds.), Elsevier/North Holland Biomedical Press, Amsterdam, pp. 157-159 (1980)) described plants regenerated from 5-MT resistant N. tabacum suspension cultures. While the cultures expressed an anthranilate synthase enzyme that was less sensitive to feedback inhibition by tryptophan and also exhibited an increased level of free tryptophan (approximately 25-fold), the leaves of the regenerated plants did not express the altered form of the enzyme and did not form roots in medium containing 5-MT. The resistance trait was, however, expressed in callus derived from the regenerated plant. Thus it appears to be difficult to obtain expression of the 5-MT resistance phenotype in tobacco plants derived from 5-MT resistant cells selected in culture.
Finally, Ranch et al. (Plant Physiol., 71, 136 (1983)) selected for 5-MT resistance in cell cultures of Datura innoxia, a dicot weed, and reported that the resistant cell cultures contained increased tryptophan levels (8 to 30 times higher than the wild type level) and an anthranilate synthase with less sensitivity to tryptophan feedback inhibition. Regenerated plants were also resistant to 5-MT, contained an altered anthranilate synthase, and had a greater concentration of free tryptophan (4 to 44 times) in the leaves than in the leaves of the control plants. In contrast to the studies with N. tabacum, where the altered enzyme was not expressed in plants regenerated from resistant cell lines, these results indicated that the amino acid overproduction phenotype could be selected at the cellular level and expressed in whole plants regenerated from the selected cells in Datura innoxia. 
Although it is possible to select for 5-MT resistance in certain cell cultures and plants, this characteristic does not necessarily correlate with the overproduction of free tryptophan in whole plants. Additionally, plants regenerated from 5-MT resistant lines frequently do not express an altered form of the enzyme. Nor is it predictable that this characteristic will be stable over a period of time and will be passed along as a heritable trait.
Thus, there is a need to increase the tryptophan content of plants and/or provide plants that are resistant to growth inhibitory amounts of tryptophan or an analog thereof.
The present invention provides an isolated and purified DNA molecule comprising a DNA segment encoding an anthranilate synthase (AS) substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan such as those discussed hereinbelow. A preferred embodiment of the invention is a plant anthranilate synthase which is substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan. Another embodiment of the invention is an isolated and purified DNA molecule comprising a DNA segment encoding a maize anthranilate synthase. A DNA molecule of the invention can further comprise an amino terminal plant chloroplast transit peptide sequence operably linked to the anthranilate synthase coding sequence.
Another embodiment of the invention provides a method for conferring tolerance to an amino acid analog of tryptophan and/or altering the tryptophan content of a plant, plant tissue, plant part, or plant cell by introducing and expressing a DNA segment encoding an anthranilate synthase in a plant cell. Also provided is a method of molecularly detecting an anthranilate synthase that is substantially tolerant to inhibition by an amino acid analog of tryptophan comprising identification of a restriction enzyme site polymorphism in the gene encoding the anthranilate synthase. The method comprises mixing DNA isolated from a culture, organism, or a portion of an organism, with an amount of NlaIII restriction endonuclease, and determining whether the restriction endonuclease cleaves the DNA within a portion of the DNA that encodes an anthranilate synthase.
The method of imparting tolerance to an amino acid analog of tryptophan to a plant, plant cell, plant part or plant tissue includes the steps of introducing a preselected DNA segment encoding an anthranilate synthase that is substantially tolerant to inhibition by the tryptophan analog or free L-tryptophan and that is operably linked to a promoter functional in a plant cell, into cells of a susceptible plant. The transformed plant cells are then regenerated to provide a differentiated fertile plant. The promoter can be an inducible or tissue specific promoter. The functional linkage of a promoter to the DNA segment results in an expression cassette. Other transcription or translation regulatory elements, e.g., enhancers or terminators, can also be functionally linked to the DNA segment. Expression of the DNA segment yields an amount of anthranilate synthase effective to render the plant, plant part, plant cell, or plant tissue substantially tolerant to an amount of an amino acid analog of tryptophan or free L-tryptophan that inhibits the growth of a corresponding plant, plant part, plant cell or plant tissue without the DNA segment (xe2x80x9cuntransformedxe2x80x9d material).
Once transformed plant cells exhibiting tolerance are obtained, transgenic plants can then be regenerated therefrom, and evaluated for stability of the inheritance of the resistance or tolerance trait, that is, whether the resistance or tolerance trait is transmitted to progeny. Thus, another embodiment of the invention is a transformed plant that is substantially tolerant to an amino acid analog of tryptophan. The cells of the transformed plant comprise a native anthranilate synthase gene and a DNA segment encoding an exogenous anthranilate synthase. The expression of the exogenous anthranilate synthase in the cells of the plant confer tolerance to the plant to an amount of an amino acid analog of tryptophan or free L-tryptophan that inhibits the activity of the native anthranilate synthase.
The transmission of the resistance or tolerance trait can be evaluated at a molecular level, e.g., Southern or Northern blot analysis, PCR-based methodologies, or the biochemical or immunological detection of anthranilate synthase, or by phenotypic analyses, i.e., whether transformed progeny can grow in the presence of an amount of an amino acid analog of tryptophan or free L-tryptophan that inhibits the growth of an untransformed plant. Also provided is a transformed plant which is substantially tolerant to growth inhibition by an amino acid analog of tryptophan or free L-tryptophan, as well as a seed therefrom.
The invention also provides a method for altering, preferably increasing, the tryptophan content in a plant. The method comprises introducing a DNA segment comprising a gene encoding an anthanilate synthase substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan, wherein the segment is operably linked to a promoter functional in a plant cell, into the cells of a plant and then expressing the gene in an amount effective to alter the tryptophan content of the plant cell. Thus, another embodiment of the invention is a transformed plant having an altered cellular tryptophan content.
In a preferred embodiment of the invention, transformed cells exhibiting about a 1.1- to 50-fold increase in total tryptophan content are selected for and used to generate transgenic plants, plant parts and seeds exhibiting a substantial increase in tryptophan content A substantial increase in tryptophan content is determined with respect to the tryptophan content normally present in the untransformed plant, plant part, e.g., leaves or fruit, or seed, and can range from about a 1.1 to a 50-fold increase over that present in the corresponding untransformed plant.
Also provided is a fertile transgenic Zea mays plant comprising a DNA segment encoding an anthranilate synthase that is substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan, operably linked to a promoter. The expression of this DNA segment in the transgenic Zea mays plant results in levels of free L-tryptophan in the cells of the transgenic plant that are substantially increased above the levels of free L-tryptophan in the cells of a Zea mays plant which only differ from the cells of the transgenic Zea mays plant by the absence of the DNA segment. This DNA segment is transmitted through a complete normal sexual cycle of the plant to its progeny and to further generations.
The invention also provides for a method of producing anthranilate synthase in a host cell. The method includes the steps of introducing an expression cassette comprising a DNA segment encoding an anthranilate synthase into a host cell and expressing the DNA segment in the host cell so as to yield anthranilate synthase. An expression cassette preferably includes transcription and translation regulatory elements, e.g., a promoter, functional in host cell, either of eukaryotic or prokaryotic origin. Preferably, the expression cassette is introduced into a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as a yeast or insect cell, that is known to be useful for production of recombinant proteins. Recombinantly produced anthranilate synthase can then be used to identify other agents that bind to and inhibit anthranilate synthase.
The present invention also provides an isolated and purified DNA molecule of at least seven nucleotide bases which hybridizes under high stringency conditions to a DNA molecule comprising a DNA segment encoding an anthranilate synthase substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan, such as a plant anthanilate synthase. Also provided is a hybridization probe comprising an isolated and purified DNA segment of at least seven nucleotide bases, which is detectably labeled or which can bind to a detectable label, which DNA segment hybridizes under high stringency conditions to the non-coding strand of a DNA molecule comprising a DNA segment encoding an anthranilate synthase, such as a plant anthranilate synthase, substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan, or a maize anthranilate synthase. High stringency conditions are defined as: hybridization at 65xc2x0 C. for at least 16 hours in 5xc3x97SSC, 1xc3x97Denhardt""s solution, 50 mM Tris-HCl, pH 8, 0.2% SDS, 10 mM EDTA, 0.1 mg/ml salmon sperm DNA, followed by washing twice for 5 minutes in 2xc3x97SSC, 0.5% SDS at 25xc2x0 C., once for 10 minutes in 0.2xc3x97SSC, 0.1% SDS at 25xc2x0 C. and twice for 30 minutes in 0.2xc3x97SSC, 0.1% SDS at 56xc2x0 C.
The present invention also provides a method of introducing an exogenous anthranilate synthase gene into a host cell comprising transforming host cells in vitro with an expression cassette comprising a DNA segment encoding an anthranilate synthase, operably linked to a promoter functional in the host cell, expanding the transformed host cells in vitro, and identifying a transformed host cell which expresses the anthranilate synthase encoded by the DNA segment. A preferred embodiment of the invention is method of introducing an exogenous anthranilate synthase gene into plant cells. A more preferred embodiment of the invention is a method wherein the transformed plant cells can be regenerated into a differentiated dicot or monocot plant.
Another embodiment of the invention is a method of selecting transformed plant cells. The method comprises introducing a preselected DNA segment into a plant cell to yield a transformed plant cell. The DNA segment encodes an anthranilate synthase which is substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan. The transformed cell is cultured in an amount of free L-tryptophan or an amino acid analog of tryptophan that inhibits the growth of a plant cell which does not contain the preselected DNA segment. A preferred embodiment of the invention is a DNA segment encoding a plant anthranilate synthase. Another preferred embodiment of the invention is a method of selecting transformed plant cells comprising introducing a preselected DNA segment into a plant cell to yield a transformed plant cell. The DNA segment encodes a chloroplast transit peptide operably linked to an anthranilate synthase which is substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan.
Also provided is an isolated and purified DNA molecule comprising a DNA segment encoding transcription regulatory elements for a plant anthranilate synthase gene.
Further provided is an isolated and purified DNA molecule comprising a DNA segment encoding a beta subunit of an anthranilate synthase.
The term xe2x80x9cconsists essentially ofxe2x80x9d as used with respect to the present DNA molecules, sequences or segments is defined to mean that a major portion of the nucleotide sequence encodes an anthranilate synthase, optionally operably linked to a chloroplast transit peptide, and that nucleotide sequences are not present which encode proteins other than an anthranilate synthase, optionally operably linked to a chloroplast transit peptide.
As used herein, xe2x80x9can amino acid analog of tryptophanxe2x80x9d is an amino acid analog of an intermediate in the tryptophan biosynthetic pathway or an amino acid analog of tryptophan. These analogs include, but are not limited to, 6-methylanthranilate, 5-methyltryptophan, 4-methyltryptophan, 5-fluorotryptophan, 5-hydroxytryptophan, 7-azatryptophan, 3xcex2-indoleacrylic acid, 3-methylanthranilic acid, and the like.
As used herein, xe2x80x9csubstantially increasedxe2x80x9d or xe2x80x9celevatedxe2x80x9d levels of free L-tryptophan in a plant cell, plant tissue, or plant are levels that are about 1.1 to 50 times, preferably about 2 to 20 times, and more preferably about 3-10 times, the levels found in an untransformed plant cell, plant tissue, or plant, i.e., one where the genome has not been altered by the presence of an exogenous gene. For example, the levels of free L-tryptophan in a transformed plant are compared with those in an untransformed plant. In the alternative, the levels of free L-tryptophan in a homozygous backcross converted inbred transformed plant are compared to the levels in a recurrent inbred plant. A homozygous backcross converted inbred transformed plant is a transformed plant which has been repeatedly crossed to the recurrent inbred parent until the transformed plant is substantially isogenic with the recurrent inbred parent except for the presence of an introduced transgene, and is then is self-pollinated (selfed) at least once.
As used herein, xe2x80x9csubstantially isogenicxe2x80x9d means that the genomic DNA content of a homozygous backcross converted inbred transformed plant is at least about 92%, preferably at least about 98%, and most preferably at least about 99%, identical to the genomic DNA content of a recurrent inbred parent of the transformed plant.
As used herein, a plant cell, plant tissue or plant that is xe2x80x9csubstantially resistant or tolerant to inhibition by free L-tryptophan or an amino acid analog of tryptophanxe2x80x9d is a plant cell, plant tissue, or plant that grows in an amount of tryptophan or an amino acid analog of tryptophan that normally inhibits growth of the untransformed plant cell, plant tissue, or plant, as determined by methodologies known to the art. For example, a homozygous backcross converted inbred plant transformed with a DNA molecule that encodes an anthranilate synthase that is substantially resistant or tolerant to inhibition by free L-tryptophan or an amino acid analog of tryptophan grows in an amount of tryptophan or an amino acid analog of tryptophan that inhibits the growth of the corresponding, i.e., substantially isogenic, recurrent inbred plant.
As used herein, an anthranilate synthase that is xe2x80x9csubstantially resistant or tolerant to inhibition by free L-tryptophan or an amino acid analog of tryptophanxe2x80x9d is an anthranilate synthase that is not inhibited by an amount of free L-tryptophan or an amino acid analog of tryptophan that normally inhibits the corresponding xe2x80x9cwild-typexe2x80x9d or native anthranilate synthase of the species.
As used herein, xe2x80x9ccells of a susceptible plantxe2x80x9d are cells which are sensitive to growth inhibition by free L-tryptophan or to an amino acid analog of tryptophan. For example, plant cells from the C28 maize cell line are not susceptible to growth inhibition by 5-methyltryptophan at levels of 5-methyltryptophan that inhibit the growth of many other maize cell lines, such as those obtained from the inbred line H99 or from hybrids such as A188xc3x97B73 (Miao et al., Plant Cell, Tissue and Organ Culture, 14, 3 (1988)). Thus, C28 cells are not cells of a susceptible plant.
As used herein, an xe2x80x9cexogenousxe2x80x9d anthranilate is an anthranilate synthase that is encoded by a DNA sequence that has been isolated from a cell, purified, and amplified.
As used herein, a xe2x80x9cnativexe2x80x9d gene means a gene that has not been manipulated in vitro, i.e., has not been isolated, purified, and amplified.
As used herein, xe2x80x9calteredxe2x80x9d levels of tryptophan in a transformed plant, plant tissue, or plant cell are levels which are greater than the levels found in the corresponding untransformed plant, plant tissue, or plant cells. In the alternative, altered levels of tryptophan in a backcross converted inbred transformed plant are greater than the levels found in the corresponding recurrent inbred plant.